Non-small cell lung cancers (NSCLCs), particularly those with activating KRAS mutations, are often unresponsive to targeted agents and have a poor prognosis. However, immunotherapeutic approaches, particularly PD-1/PD-L1 pathway blockade, have recently improved the treatment of these cancers, supporting the premise that evasion of immune destruction contributes to NSCLC pathogenesis. MUC1-C is an oncogenic protein that is overexpressed in >80% of NSCLCs and is linked to multiple immune signaling pathways. Work supported by this grant has demonstrated that EGFR mutant NSCLCs activate the PD-1/PD-L1 pathway and increase production of proinflammatory cytokines. We have also shown that MUC1-C promotes (i) oncogenic signaling in NSCLCs via EGFR activation, and (ii) confers EMT, self-renewal and tumorigenicity in KRAS mutant NSCLCs. Based on these findings, we have generated novel antibodies against the non-shed MUC1-C extracellular domain and inhibitors of the cytoplasmic domain for therapeutic targeting of MUC1-C in NSCLC cells. Our preliminary observations indicate that targeting MUC1-C downregulates PD-L1 expression in EGFR and KRAS mutant NSCLC cells. We have also found that MUC1-C deficiency is associated with recruitment of tumor-associated macrophages and alterations in the infiltrating T-cell phenotype. Our hypothesis is that MUC1-C plays an important role in evading immune destruction, which will be addressed in studies of (i) human NSCLC cell lines, (ii) genetically engineered mouse models, and (iii) primary NSCLC tumors. The Specific Aims are: (1) To investigate the role of MUC1-C in PD-L1 pathway activation in EGFR and KRAS mutant NSCLC cells, (2) To define the role of MUC1-C in recruitment and function of macrophages and T-cells within the tumor microenvironment of KRAS and EGFR mutant NSCLC, (3) To evaluate the effects of targeting MUC1-C to circumvent immune evasion in mutant EGFR and KRAS NSCL tumors, and (4) To assess fresh resected/biopsied lung cancer specimens and correlate MUC1 expression with T-cell and other immune cell infiltration, immune checkpoint gene expression, and cytokine secretion in the tumor microenvironment.